Purification of the Moloney and Rauscher Murine Leukemia Viruses by Use of Zonal Ultracentrifuge Systems

The B-IV and B-IX zonal ultracentrifuge rotors were applied to the concentration and purification of the Moloney and Rauscher murine leukemia viruses from large volumes of infected tissue culture fluids and animal materials. Potassium tartrate, potassium citrate and sucrose gradients were used to obtain viral concentrates from the density 1.16 to 1.18 zone. Proteolytic enzyme digestion of tissue culture preparations prior to zonal ultracentrifuge processing was effective in releasing virus from cell debris and producing highly purified, though nonleukemogenic, viral concentrates. Infected Rauscher mouse plasma was processed to give highly purified infectious virus fractions. A single centrifugation of crude Rauscher mouse spleen homogenates resulted in partially purified infectious concentrates with high virus particle counts.     

Large-Scale Purification of Ribonucleic Acid Tumor Viruses by Use of Continuous-Flow Density Gradient Centrifugation

This report describes a 200- to 300-fold increase in the quantity of ribonucleic acud tumor virus particles previously isolated at one time by zonal centrifugation.     

Isolation of Relaxing Particles from Rat Skeletal Muscles in Zonal Centrifuges

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s _20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.     

Rapid Purification of 70S RNA from Media of Cells Producing RNA Tumor Viruses

Media from cells producing RNA tumor viruses, when treated with sodium dodecyl sulfate and polyvinyl sulfate, yield 70S RNA as the major species binding oligo(dT)-cellulose. The procedure described for purifying 70S RNA requires no special equipment and is suitable for rapidly processing large quantities of media or for purifying RNA from commercially available virus, with a 5- to 10-fold higher yield than was obtained using existing methods.     

Purification of Large Amounts of Murine Ribonucleic Acid Tumor Viruses Produced in Roller Bottle Cultures

Murine ribonucleic acid tumor viruses and C-type virus particles are produced in relatively large quantities in roller bottle cultures. The viruses present in large volumes of culture fluids can be purified by a simple two-step procedure involving polyethylene glycol precipitation and equilibrium centrifugation in sucrose density gradients.     

Purification, Characterization, and Comparison of the DNA Polymerases from Two Primate RNA Tumor Viruses

The DNA polymerase from the Mason-Pfizer monkey virus (M-PMV), an RNA tumor virus not typical type-C or type-B, has been purified a thousand-fold over the original crude viral suspension. This purified enzyme is compared to a similarly purified DNA polymerase from the primate woolly monkey virus, a type-C virus. The two enzymes have similar template specificities but differ in their requirements for optimum activity. Both DNA polymerases have a pH optimum of 7.3 in Tris buffer. M-PMV enzyme has maximum activity with 5 mM Mg(2+) and 40 mM potassium chloride, whereas the woolly monkey virus optima are 100 mM potassium chloride with 0.8 mM Mn(2+). The apparent molecular weight of the M-PMV enzyme is approximately 110,000, whereas the woolly monkey virus polymerase is approximately 70,000. The biochemical properties of these two enzymes were also compared to a similarly purified enzyme from a type-C virus from a lower mammal (Rauscher murine leukemia virus). The results show that more similarity exists between the DNA polymerases from viruses of the same type (type-C), than between the polymerases from viruses of different types but from closely related species.     

大规模区带离心纯化Vero细胞乙脑疫苗

本文报道一种适合疫苗生产的大规模纯化Ｖｅｒｏ细胞乙脑疫苗的方法。原疫苗经适当浓缩和去除ＤＮＡ处理后,用不连续蔗糖梯度（３６％和６０％）。３２６００ｇ,速率区带离心４ｈ。纯化后疫苗的效力比中国参考疫苗高出６倍以上,补体结合抗原比中国参考疫苗高４～８倍。总蛋白含量低于３０μｇ／ｍＬ,牛血清含量降至０．５μｇ／ｍＬ以下,细胞残余ＤＮＡ低于１００ｐｇ／０．５ｍＬ。用此法连续制备三批纯化疫苗,其纯度和效力均高于日本鼠脑纯化疫苗。此法对于制备其它纯化Ｖｅｒｏ细胞疫苗也具有一定的参考意义。     

10. Purification of Viruses by Cenlrifugation in Gradients of Inert Substances

ABSTRACT

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in “self formed” caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Centrifugation in gradients of inert substances is useful for the separation of components in a mixture¹. There are two principles involved in this type of separation. One, termed reorienting gradient centrifugation (reograd) relies on the differences in masses or, better still on the sedimentation coefficients of the different components in the mixture and the second, termed isopycnic centrifugation², on the densities or specific gravities of the different entities.     

Use of Yeast Populations Fractionated by Zonal Centrifugation to Study the Cell Cycle

Zonal centrifugation in a sucrose density gradient was used to separate yeast cells primarily by size and thus by age in the cell cycle. This approach provides an alternative to synchronous growth for examining the properties of cells at different stages in the cell cycle.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

肿瘤病毒诱导细胞异常增殖作用机制的研究进展

肿瘤是当前危害人类健康最严重的疾病,它以细胞增殖为主要病变特征.自从1911年Rous发现鸡肉瘤病毒可以使鸡发生白血病,人们就将病毒与肿瘤联系起来.本文就肿瘤病毒诱导细胞异常增殖作用机制方面分两部分进行阐述.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B(77)), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB(77)), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.     

Properties of Noninfectious and Transforming Viruses Released by Murine Sarcoma Virus-Induced Hamster Tumor Cells

The cell culture lines HTG2 and HTG3 were established from a transplantable hamster tumor induced by a murine sarcoma virus (strain Gz-MSV) after 17 and 60 in vivo passages, respectively. The viruses released by these two cell lines markedly differ in morphology, antigenic composition, infectivity, transforming ability, and enzymatic activity. HTG2 virions contained the sarcoma genome but were noninfectious for mouse and hamster cells (S+H-virus). HTG3 virions transformed hamster but not mouse cells. Whereas HTG2 cells and its virus contained murine type C virus gs-1 antigen, all HTG3 clonal lines expressed both murine and hamster type C virus gs-1 antigens. The RNA-dependent DNA polymerase activity of HTG2 virus was very low, whereas that of HTG3 virus was relatively high. HTG2 virions contained electron-lucent centers only. HTG3 virus consisted of the expected mixture of virions with electron-dense and electron-lucent centers. Many broken or incomplete virions were present in both viruses. HTG2 virus is a noninfectious "defective" sarcoma virus without detectable helper virus. Data obtained in these experiments suggest that HTG3 virus is a hamster type C virus pseudotype of Gz-MSV (Gz-MSV [HaLV]). The genome of Gz-MSV is capable of antigenic expression in heterologous cells and in the presence of heterologous viruses. Attempts to chemically activate hamster type C virus (HaLV) from HTG2 cells were unsuccessful. The HTG1 cell culture line, established from another Gz-MSV-induced hamster tumor, initially released a virus indistinguishable from the HTG2 virus. After in vitro passage, spontaneous activation of HaLV occurred in HTG1 cells, and the resultant Gz-MSV (HaLV) had properties similar to those of the HTG3 virus.     

Role of Mitochondria in the Production of RNA-Containing Tumor Viruses

The role of mitochondria in the reproduction of RNA-containing tumor viruses was examined by using ethidium bromide (EB) to induce degenerative effects in mitochondria. The effects of EB in murine and avian cells were monitored by electron microscopy. Chronically infected mouse (JLS-V5) cells, in which extensive mitochondrial changes were induced, continued to produce murine leukemia virus. Also, complete reproductive cycles of Rous sarcoma virus (RSV) occurred in newly infected chicken embryo cells exposed to EB. Morphological transformation characteristic of infection of chicken embryo cells by RSV occurred in cells which contained induced aberrant mitochondria. The results demonstrate that mitochondria play a relatively minor role, if any, in the reproduction of RNA-containing tumor viruses.     

Synchronization of Escherichia coli by Zonal Centrifugation

A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.     

Use of Plant Viruses for Production of Plant-Derived Vaccines

Plants produce appropriately folded, post-translationally processed proteins that, as antigens, elicit efficacious immune responses in preclinical animal models and antigen-specific responses in humans. Plant-produced vaccine candidates have been produced using transgenic technologies and the utilization of plant viruses for the transient protein expression. The later approach has numerous advantages in recombinant protein production, including rapid protein expression and higher yields of antigenic proteins. In some cases, plant viruses are “decorated” with human or animal antigens from pathogens to form chimeric virus particles (CVPs). Immunization of animals with CVPs induces specific and often efficacious immune responses. While there are no plant-produced vaccines commercially available, the diversity and effectiveness of the products presently in development coupled with production advantages, including, reduced cost of production, the rapid scale-up capabilities, and the safety of the final product, should encourage continued investment and progress through clinical testing.